Hypermethylation of promoter CpG islands is an important mechanism to silence the expression of many tumor suppressors, DNA repair, and other genes in cancer. The long-term goal of this project is to study the inhibition and reversal of this process by dietary polyphenols for the purpose of prevention and treatment of cancer. In preliminary studies, we observed that treatment of human esophageal cancer KYSE 510 cells with the tea polyphenol EGCG caused the reversal of hypermethylation of RARbeta, p16, MGMT, and hMLH1 and the regaining of gene expression. EGCG also inhibited DNMT activity in nuclear extracts. This property of EGCG and related compounds could be explored for cancer chemoprevention and therapy. Our hypothesis is that EGCG and some other dietary polyphenols can inhibit DNMT, prevent or reverse the gene silencing caused by hypermethylation, and contribute to the inhibition of carcinogenesis, In this proposal, we plan to test this hypothesis with the following Specific Aims: 1. To quantify the demethylation and reactivation of hypermethylation-silenced genes (MSGs) such as RARbeta, p16, MGMT, and hMLH1 by EGCG, characterize the pattern of CpG demethylation, and determine the sustainability of the reactivated gene expression. 2. To elucidate the mechanisms by which EGCG inhibits DNMT activity and DNA hypermethylation. The leading hypothesis is that inhibition of DNMT1 by EGCG causes demethylation. Other mechanisms such as those involving AdoMet levels and other methyltransferases, will also be examined. 3. To establish the above observed effects as general phenomena by determining the spectrum of MSGs that are reactivated by EGCG (using methylation microarrays), examining the effects of EGCG on other cell lines (oral, colon, and prostate cancers), and investigating the effects of other dietary polyphenols. 4. To determine the possible synergistic effects in the reactivation of MSGs when EGCG is used in combination with a HDAC inhibitor (butyrate or TSA) or retinoic acid. Possible effects on global hypomethylation will be analyzed. 5. To determine whether EGCG, alone or in combination with other agents, can prevent hypermethylation of genes and tumor development in the intestinal tumorigenesis model in the Min mice.